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Mapping the Toxoplasma gondii cytosolic interactome by crosslinking mass spectrometry and machine learning

ABSTRACT: Protein–protein interactions (PPIs) are fundamental to cellular processes and pathogen biology, yet remain undercharacterized in the human parasite Toxoplasma gondii. Here, we apply crosslinking mass spectrometry (XL-MS) to map the cytosolic interactome of T. gondii tachyzoites at proteome scale. Using an optimized workflow that combines MS-cleavable and non-cleavable crosslinker searches, we identified 391 PPIs and over 5,000 residue-residue contacts, including known complexes such as the ribosome and proteasome, as well as novel interactions among dense granule proteins. Structural mapping confirmed the spatial plausibility of crosslinks, while detection of homo-dimers and transient interactions highlighted the sensitivity of our approach. We further employed machine learning using LightGBM to rescue lower-confidence interactions by integrating large-scale biological data including gene expression, subcellular localization, and known functional associations. The model achieved 98.7% accuracy and enabled expanded interactome reconstruction. This study establishes XL-MS as a powerful tool for characterizing PPIs in parasites and provides a foundational interactome for T. gondii, with implications for understanding parasite biology and host-pathogen interactions.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Toxoplasma Gondii Type I

SUBMITTER: Simone Sidoli

LAB HEAD: Simone Sidoli

PROVIDER: PXD062479 | Pride | 2025-07-26

REPOSITORIES: Pride

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	20231014_Jenny_Tadakimi_Toxo_XL_s1_1_s1_5_7.raw	Raw
	20231014_Jenny_Tadakimi_Toxo_XL_s1_2_s1_6_8.raw	Raw
	20231014_Jenny_Tadakimi_Toxo_XL_s1_3_s1_7_6.raw	Raw
	20231014_Jenny_Tadakimi_Toxo_XL_s1_4_s1_8_5.raw	Raw
	20231014_Jenny_Tadakimi_Toxo_XL_s1_FT_9.raw	Raw

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